Edinburgh Napier University

  Background: The Safe and Sustainable by Design (SSbD) concept facilitates the design of safer and more sustainable chemicals and materials and is a crucial approach towards reaching the goals set out in the European Green Deal. It is critical that suitable guidance is provided on how to use new approach methodologies (NAMs) to fill hazard data gaps for nanomaterials (NMs) to facilitate SSbD decisions. Here, we showcase a nano-specific in vitro SSbD case study. The five colloidal silica nanoforms (SiO2-NFs) under investigation in this study are surface modified with varying amounts of glycerolpropyl-organosilane groups. In this study, we use a simple yet comprehensive in vitro test battery along with thorough particle characterization to investigate the effect of surface silanization on in vitro toxicity to inform SSbD decisions. Results: Cytotoxic, pro-inflammatory and oxidative stress responses in A549, dTHP-1, and BEAS-2B cells after exposure to SiO2-NFs submerged and at the air-liquid interface (ALI) decreased with increasing silane surface modification. None of the SiO2-NFs showed surface reactivity or haemolytic potential. Deposition assessment using inductively coupled plasma – optical emission spectrometry (ICP-OES) revealed that increasing silane surface modification decreased particle settling. The two SiO2-NFs with the highest amount of surface silanization did not reach the cells in a submerged exposure setting, and they were therefore only tested at the ALI. Identical dose-response curves were observed for both the submerged testing and testing at the ALI for the SiO2-NFs without and with low/intermediate surface functionalization, again showing a decrease in effects with increasing surface functionalization. Conclusion: We show that in vitro toxicity assays provide valuable information for SSbD decision making. In vitro cytotoxic, pro-inflammatory and oxidative stress responses can be reduced with increasing surface silane functionalization. The reduced deposition efficiency with increasing silane functionalization, however, highlights that thorough characterization of particle behaviour in cell culture medium should always be performed for SSbD hazard testing. The amount of silane required to reduce toxicity is important information for the future production of safer SiO2-NFs and nano-enabled products. Exposure, functionality, and sustainability remain to be investigated to draw full SSbD conclusions.